Am working on Bacillus thuringiensis, I need to purify the Parasporal Crystals for Bioassay works, any efficient way to isolate protein crystals that should exclude spores ?.
For preparation of parasporal crystals on a somewhat larger scale, you might want to consider density gradient centrifugation. The most effective methods, I believe, use a diatrizoate meglumine and diatrizoate sodium solution for the gradient. It is marketed as a high osmolarity contrast agent for medical imaging techniques and is available under various brand names. For details on the method, see Sharpe et all, Appl Microbiol. 1975 Dec;30(6):1052-3
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC376592/
Article Separation of Spores and Parasporal Crystals of Bacillus thu...
I'm glad to see my 1975 paper using renografin is still remembered. I was a post-doc then. We used renografin because that was what Wise and Halvorson (JB 94 2075 1967) had used to isolate spores of altered density which no longer made DPA. It was important that the cells still could be viable, and renografin was considered benign. However, renografin is expensive. When I got my own lab we switched to sodium bromide (Ang and Nickerson AEM 36 625-626 1978), which had the added advantage (Nickerson and Swanson, Eur J Appl Microbiol Biotech 13 213-215 1981) that harvesting the crystals from 30% NaBr stripped the sporulation related proteases off the crystals. Otherwise the crystal proteins were hard to study because they were being hydrolyzed as soon as they were solubilized. We continued to use NaBr purified crystals throughout our work for the next 25 years. I still have several 100 pound drums of NaBr.
Thanks, Ken. :) Years ago I talked someone in the radiology department at our university hospital into giving me a case of Renocal that was about to expire. I've used it since then for small scale purification of Bt crystals as well as spores from B. subtilis and B. atrophaeus. Your method has worked great for me. I can see that for large scale preps it would get expensive. It's good to know about the advantages of NaBr.
I had tried with Hexane method, but couldn't purify 100% Crystals. i tried as they had mentioned in the literature and as i keep changing the hexane after centrifugation, the froth used to settle at the walls of the tube and some times the pellet was used to wash with supernatent .
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